Telomeres are repeated hexanucleotide sequences at the ends of linear chromosomes, which serve to protect them from recognition as chromosomal breaks; furthermore, the asymmetric replication of DNA would lead inevitably to a loss of genetic material, and telomerase, an enzymatic complex that adds telomeric sequences at mitosis, functions to maintain genomic integrity. Telomerase deficiency manifests with short telomeres and loss of both enzymatic activity: its consequences can be measured in vitro and in vivo. Mutations in DKC1 and in TERC (the RNA template subunit of the complex) are etiologic in some cases of dyskeratosis congenita, a constitutional form of aplastic anemia. Mutations in TERT (encoding telomerase, the rate limiting enzymatic component of the complex) occur in apparently acquired aplastic anemia and other diseases. Heterozygous mutations in TERT lead to defective telomere repair and short telomeres due to a mechanism of haploinsufficiency. Male hormones, long used to treat aplastic anemia, act by up regulating TERT transcription and telomerase activity, including in lymphocytes and hematopoietic progenitor cells. While critical telomere shortening often leads to either cell senescence or apoptosis, occasional cells become anneuploid due to end-to-end fusion of chromosomes. Thus, telomere attrition is a mechanism for oncogenesis. Telomere length of leukocyte is now measured routinely in our CLIA laboratory by gene amplification using robotic methodology provided by a Quiagen Quiagility and Rotor GeneQ; high throughput analysis is useful both for research and in the clinic, and our procedure is certified for patient data. Measurement of clinical samples is required for the adequate diagnosis of aplastic anemia and is predictive of late events after treatment with immunosuppression, and probably in other clinical circumstances. We now have established single telomere length analysis (STELA), which relies on amplification using chromosome specific sub-telomeric DNA sequence to detect critical telomere shortening in individual chromosomes. We utilize flow-FISH, a flow cytometry assay that allows measurement of telomere length individual cells in suspension. As reported last year, we analyzed telomere attrition in a subgroup of patients with aplastic anemia who ultimately developed monosomy 7, the dominant cytogenetic abnormality in clonal evolution from aplastic anemia to myelodysplastic syndrome/acute myeloid leukemia. Accelerated telomere attrition was insistently present in these patients, but only a few showed mutations in MDS/AML candidate genes. We are incorporating telomere testing at diagnosis and at three and six months post treatment prospectively to assess patients in our clinic for prognosis in general and for risk of development of myeloid malignancy. Of note, in patients who evolve to MDS/AML on eltrombopag therapy, there was also little evidence of candidate gene mutation in serial samples; serial telomere measurements are also being performed in these cases. We are utilizing a combination of deep clinical phenotyping and machine learning to model telomere disease in the context of bone marrow failure syndromes in general as well as to characterize genotype-phenotype relationships. Over 150 patients with a putative diagnosis of telomeropathy, with or without documented gene mutations, have been compared to a similar number of patients with moderate and severe aplastic anemia. Among telomere disease patients, most (66) have TERT mutations, followed by TERC (25), and unidentified mutations (40). Bothe two and three class random forest analyses have been applied. A large number of clinical and laboratory parameters are available in these patients. A family or personal history of lung disease was a major discriminator between TERC and TERT telomere disease, and a relatively limited cassette of clinical features appears to be predictive with high sensitivity and specificity. Early greying was more characteristic of TERT mutant patients. TERC was distinguishable from unidentified mutations while patients with unidentified mutations tend to cluster with those who have TERT deficiency. For analysis in the context of bone marrow failure, random forest disclosed clustering of subgroups of patients, distinct from those with moderate and severe aplastic anemia; characterization of these subgroups by clinical parameters in genotype is in progress. The major predictors of telomere disease versus aplastic anemia in general was family or personal history of androgen therapy and a personal history of early greying of the hair and lung diseases. Application of machine learning in this context is unusual and should be generally applicable in other diseases in which subsets of patients have clearly defined molecular mechanisms. Finally, we have completed and submitted for publication our interventional trial of danazol, a synthetic androgen, in patients with suspected telomere disease, defined as short telomeres in the context of an appropriate clinical syndrome, with or without documented mutation in TERT, TERC, or other telomere repair complex gene or shelterin protein gene. A total of 27 patients were enrolled to receive danazol at maximal doses for two years, with the primary endpoint being drug safety and alteration in the telomere attrition rate. Adverse effects were anticipated and observed, mainly related to changes in lipid profiles, elevated liver enzymes, and muscle cramps. Nevertheless, danazol was generally well tolerated. Remarkably, accelerated telomere attrition at a rate of 156 base pair loss annually was reversed by administration of an androgen, leading to an average gain in telomere length of 175 base pairs at six months and 360 base pairs at 12 months as compared to baseline. These data were generated using q-PCR and confirmed when patient samples were available by flow-FISH. The hematologic response rate was high, 70% at three months and 63% at six months; for those patients able to complete two years on study, 83% had improved blood counts. This study represents the first favorable modulation of telomere length in humans. Although not primary or secondary endpoints, there were also improvements in pulmonary function, as measured by CO diffusion capacity, and stabilization of liver fibrosis on imaging. We anticipate new protocols examining lower doses of androgens in telomeropathy patients as well as the application of androgen therapy to patients with predominantly lung and liver disease with underlying telomere gene mutations.